SIV Evolutionary Dynamics in Cynomolgus Macaques during SIV-Mycobacterium tuberculosis Co-Infection.

Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6-9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. SIV diversity was highest in the thoracic lymph nodes compared to other sites, suggesting that lymphatic drainage from the lungs in co-infected animals provides an advantageous environment for SIV replication. This is the first assessment of SIV diversity across tissue compartments during SIV-Mtb co-infection after established Mtb latency.

High-Resolution Sequencing of Viral Populations during Early Simian Immunodeficiency Virus Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses.

Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (≤29 weeks postinfection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultradeep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing-based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies, leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep-sequencing-based fitness assay, we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

A penalized regression approach to haplotype reconstruction of viral populations arising in early HIV/SIV infection.

MOTIVATION: Next generation sequencing (NGS) has been increasingly applied to characterize viral evolution during HIV and SIV infections. In particular, NGS datasets sampled during the initial months of infection are characterized by relatively low levels of diversity as well as convergent evolution at multiple loci dispersed across the viral genome. Consequently, fully characterizing viral evolution from NGS datasets requires haplotype reconstruction across large regions of the viral genome. Existing haplotype reconstruction algorithms have not been developed with the particular characteristics of early HIV/SIV infection in mind, raising the possibility that better performance could be achieved through a specifically designed algorithm. RESULTS: Here, we introduce a haplotype reconstruction algorithm, RegressHaplo, specifically designed for low diversity and convergent evolution regimes. The algorithm uses a penalized regression that balances a data fitting term with a penalty term that encourages solutions with few haplotypes. The regression covariates are a large set of potential haplotypes and fitting the regression is made computationally feasible by the low diversity setting. Using simulated and in vivo datasets, we compare RegressHaplo to PredictHaplo and QuRe, two existing haplotype reconstruction algorithms. RegressHaplo performs better than these algorithms on simulated datasets with relatively low diversity levels. We suggest RegressHaplo as a novel tool for the investigation of early infection HIV/SIV datasets and, more generally, low diversity viral NGS datasets. CONTACT: sr286@georgetown.edu. AVAILABILITY AND IMPLEMENTATION: https://github.com/SLeviyang/RegressHaplo.

Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

We have constructed a single chain fragment variable (scFv) phage display library from a simian immunodeficiency virus (SIV)-infected rhesus macaque that developed unusually high-titer neutralizing antibody responses against tier-3, neutralization-resistant SIVmac239. The library was screened using trimeric (gp140) and monomeric (gp120) forms of the SIVmac239 envelope (Env) glycoprotein. We also cloned variable-heavy and variable-light (VH-VL) antibody fragments from seven previously described rhesus macaque B-cell lines (BLCLs) that produce SIV gp120-specific monoclonal antibodies (mAbs). Thirty-two gp140-specific mAbs were selected along with 20 gp120-specific ones. gp120-specific mAbs were only from the VH4 family, while gp41-specific mAbs were primarily from VH1, followed by VH4 and VH3. Rhesus macaque BLCL-derived mAbs belonged primarily to the VH4 family of antibodies followed by VH3 and a smaller number of VH1s. A preferential VH combination with Vλ light chain was observed with phage display-selected SIV Env-specific mAbs (gp120 and gp140), but not with BLCL-derived antibodies or the unpanned library. None of the tested antibodies had detectable neutralizing activity against tier-3 SIVmac239. The majority of gp120-specifc mAbs potently neutralized tier-1 SIVmac316 with 50% inhibitory concentration (IC50) values below 1 µg/ml. For gp140-specific antibodies, which were all specific for the gp41-subunit, 2 out of 11 tested neutralized SIVmac316 (IC50 of 7 and 5 µg/ml, respectively). These data suggest an order of preferential VH segment usage for SIV-specific antibodies in rhesus macaques. These antibodies will be useful in assessing the contribution of non-neutralizing antibodies to inhibition of SIV infection in vitro and in vivo.